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AQUAMUTANT

Random mutagenesis is a powerful tool for life science research. Activated AquaMutantTM can chemically modify nucleobases, and cause base substitutions during DNA replication in vitro and in vivo. By treating biological samples, such as purified DNA, plasmids, phages, virus, bacteria, yeast, cultured animal and plant cells, sperms, eggs, seeds, Arabidopsis, C. elegans, Drosophila, and Zebrafish, etc. with activated AquaMutant, researchers can create various mutant libraries. The mutant libraries may be used to investigate biological processes and disease mechanisms, screen for enzymes and antibodies with improved properties, and use directed evolution to generate mutants with desired functionalities and phenotypes

am directed evolution.png




Features 

Simpler, safer and controllable

We have made chemical mutagenesis simpler, safer and controllable. First, AquaMutant is activated to become mutagenic by mixing with the Start solution. Secondly, activated AquaMutant can be inactivated by the Stop solution after the samples have been treated for x amount of time (e.g., 4 hours), allowing you to control the mutation frequency, end the cytotoxicity and ensure reproducibility. Finally, any leftover activated AquaMutant can be destroyed by autoclave or by incubating at 75-95 C for 24 hours.

Used for both in vitro and in vivo random mutagenesis


Unlike other commercially available random mutagenesis kits, which are based on error-prone PCR to introduce mutations into PCR products, AquaMutant can introduce random mutations to not only isolated DNA fragments or intact plasmid DNA in vitro, but also DNA inside live cells and organisms in vivo.

Multiple targets

AquaMutant can mutate multiple genes concurrently in vivo. Ever wonder why the mutation and knockout of an overexpressed drug exporter did not avert drug resistance in a tumor cell. Turned out other exporters had compensated the mutated exporter. Using in vivo random mutagenesis to mutate multiple related genes could enable the alteration of complex phenotypes and the elucidation of physiopathological conditions involving multiple genes.
 
New research and discovery opportunities

Just expose millions of virus, cells or model organisms to Activated AquaMutant, you have got a mutant library, ready for finding mutants with desired properties and phenotypes. No DNA isolation, cloning and transfection is required. You could evolve phages that overcome phage-resistant bacteria; use rounds of phage random mutagenesis and phage display to improve an existing antibody drug.The research and discovery possibilities are only limited by your interest and imagination.


    Example applications of random mutagenesis

Mutate the VirusMutate the Host
Study viral infectivityIdentify cellular receptors for viral entry
Investigate viral replicationIdentify host factors supporting viral life cycle
Enhance viral antigenicityDiscover host targets for gene therapy
Create attenuated vaccineStudy viral resistance
Understand viral drug resistanceUnderstand viral pathogenesis


pUC19-BMH
In Vivo Mutagenesis. E. coli strain BMH 71-18 mutS harboring pUC19 was treated with 0, 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, and 25% of activated AquaMutant in saline at 37 C for 4 hrs. The treated bacteria were diluted with LB medium and transfered to an Amp/IPTG/XGal agar plate and incubated at 37 C for 24 hrs. All bacterial colonies were blue without being exposed to activated AquaMutant; more than 80% and 40% of bacterial colonies were white when treated with 3.1 and 1.6% of activated AquaMutant; and no bacteria had survived after being treated with >6.3% activated AquaMutant. (Note: No white colony could be generated in E. coli TOP10 strain under similar condition. Presumably TOP10 E.coli may be able to repair or remove the AquaMutant modified nucleobases or mismatches.)


Comparison
  

    Comparison of commonly used random mutagenesis methods

 

Error-prone PCR

Transposon 

AquaMutant *
Gene mutationIn vitroIn vivoIn vitro and in vivo

Knowledge of target gene

Required Not requiredNot required

DNA in vitro manipulation

Required

Required

Not required

Scope of mutation

One gene

Multiple but limited

Single to "all" genes
Phenotypes per library Limited LimitedGet what's screened for

Complex disease models

Difficult

Difficult

Mutate related genes

Directed evolution

Difficult DifficultMutate-select cycles
               
* Activated AquaMutant can be used to treat purified plasmid DNA containing the gene of interest directly to create a large mutant library without doing any cloning. Lethal mutations elsewhere in the plasmid, such as antibiotic resistance gene and origin of replication, will not be represented in the library.


Using forward genetics to decode the biology black box

Create random mutation library
!
Select mutants with the desired properties
!

Identify the mutations by whole genome sequencing
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Confirm the roles of the mutations by targeted mutagenesis






Brief Protocol 

WARNING: ACTIVATED AQUAMUTANT IS A STRONG MUTAGEN. 
Wear protective lab coat, gloves, masks and goggles to prevent skin contact or ingestion of the reagent! 

WARNING: Selection or evolution of dangerous mutants or pathogens may be dual-use research of concern (DURC).
You must seek pertinent permits and carry out DURC studies in appropriate biosafety facilities!

A. In vitro random mutagenesis

1. AquaMutant activation: Activated AquaMutant is unstable and should be prepared immediately before use. To activate AquaMutant, mix the AquaMutant solution and the Start solution at a ratio of 1:1 (e.g., 100 ul each) and incubate at 22 C for 5 min.

2. AquaMutant treatment: Suspend the DNA (restriction fragment or PCR product, 0.5-50 mg) in 1 ml of deionized water. Divide it into 8 x 100 ul aliquots in eight 1.5-ml microfuge tubes labeled #1-8. Add 100 ul of activated AquaMutant to Tube #8, mix well and then remove 100 ul to Tube #7, and so on to obtain activated AquaMutant concentrations of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50% (v/v). Incubate the samples at 37 C for 4-24 hours. (Note: To ensure a large library size and full representation, micrograms of DNA may be used in each reaction. To further simplify in vitro random mutagenesis, you may directly treat the plasmid DNA containing the gene of interest with activated AquaMutant and disregard mutations elsewhere in the plasmid for the time being). The mutation rate is dependent on AquaMutant concentration and treatment time, users may prepare a concentration series (e.g., 0.1-100% activated AquaMutant at 37 C for 4 hours) and a time series (e.g., 1, 2, 4, 8, 24 hours at 10% activated AquaMutant) to control the mutation rate for their specific projects. The DNA samples treated with different AquaMutant concentrations and exposure time may be pooled to obtain a library containing mutants with various degrees of mutations or they may be processed separately to obtain a smaller and manageable mutant library for screening.)

3. Reaction termination: Add 0.5 vol of Stop solution (with respect to the activated AquaMutant solution, e.g., for 50 ul activated AquaMutant used, add 25 ul of Stop solution) to the sample. To clean up the mutated DNA, mix the stopped reaction with an equal vol of 3 M sodium acetate (pH unadjusted, do not use acidic sodium acetate or the DNA will not precipitate), and then with an equal vol of isopropanol (e.g., for 50 ul of DNA, 50 ul of activated AquaMutant, 25 ul of Stop solution, and 125 ul of 3M NaOAc, add 250 ul of isopropanol). Incubate at 22 C (do not incubate in the freezer) for 30 min and centrifuge at 14,000 xg at 22 C for 10 min to pellet the DNA. Aspirate to remove the supernatant. Add 1 ml of 75% ethanol to the DNA pellet and flip the tube to discard the ethanol solution. Repeat the ethanol rinse once. Air-dry the DNA pellet by leaving the opened microfuge tube up-side-down on a paper towel for about 15 min. Suspend the DNA pellet in TE buffer for PCR amplification or direct cloning.


B. In vivo random mutagenesis

1. AquaMutant activation: Activated AquaMutant is unstable and should be prepared immediately before use. To activate AquaMutant, mix the AquaMutant solution and the Start solution at a ratio of 1:1 (e.g., 100 ul each) and incubate at 22 C for 5 min.

2. AquaMutant treatment: Concentrate the virus (e.g., phages) or bacteria or cells or organism to be mutated in 50 ul of saline or culture medium. Aliquot 100 ul of saline (Important: Do not use culture media for the reaction as they may contain excess amount of nucleotides and other components, which may quench the activated AquaMutant) in eight 1.5-ml microfuge tubes labeled #1-8. Add 100 ul of activated AquaMutant to Tube #8, mix well and then remove 100 ul to Tube #7, and so on to obtain activated AquaMutant concentrations of 0, 0.78, 1.56, 3.12, 6.25, 12.5, 25, and 50% (v/v). Add 1 ul of the concentrated microbes to be mutated into each tube. Incubate the samples at 37 C for 4 hours. (Note: Millions of individual cells or organisms may be needed in each reaction in order to obtain the desired mutations and phenotypes. The volumes given are for illustration only and should be adjusted appropriately for different samples, e.g., small volumes for virus but large volumes for worms. The mutation rate is dependent on AquaMutant concentration and treatment time, users may prepare a concentration series (e.g., 1 -100% activated AquaMutant at 37 C for 4 hours) and a time series (e.g., 1, 2, 4, 8, 24 hours at 10% activated AquaMutant) to control the desired mutation rate for their specific cells or organisms).

3. Reaction termination: Add 0.5 vol of Stop solution (with respect to the activated AquaMutant solution, e.g., for 50 ul activated AquaMutant used, add 25 ul of Stop solution) to the sample. Immediately pellet the treated cells by centrifugation to remove the treatment medium and resuspend the cells in fresh culture medium. Alternatively, the treated cells may be diluted directly in the culture medium without removal of the treatment medium as long as the final AquaMutant concentration is diluted to less than 1-5% (some species may be more sensitive or have stronger repair mechanism than the others). Determine the viability of the culture after 24-72 hours. Freeze down aliquots of the amplified mutant library before applying selection pressure to the remaining culture.




 Ordering Information


Product Name:    AquaMutant Kit

Product Number:   1201, 1215

Application:   In vitro and in vivo random mutagenesis. For research use only.

Size:    The kit is sufficient for the preparation of

      • 1201Varied, depending on sample volume
      • 1215: Varied, depending on sample volume

Kit Contents:   The AquaMutant Kit includes the following items

      • 1201: 1 ml each AquaMutant, Start and Stop Solutions, Instruction Manual
      • 1215: 15 ml each AquaMutant, Start and Stop Solutions, Instruction Manual

Price:  

      • 1201: $30 each
      • 1215: $299 each

Ordering:    To order, please click the "BUY" button below.

 

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MultiTarget Pharmaceuticals, LLC - All rights reserved